Method and instrument for microscopy

ABSTRACT

A microscope includes a light source that emits an illuminating light beam for illumination of a specimen, a beam splitter separating measuring light out of the illuminating light beam, and an apparatus for determining the light power level of the illuminating light beam. The apparatus for determining the light power level of the illuminating light beam receives the measuring light and includes an apparatus for simultaneous color-selective detection of the measuring light.

CROSS REFERENCE TO RELATED APPLICATIONS

This invention is a continuation of U.S. patent application Ser. No.09/881,049, filed Jun. 15, 2001, now U.S. Pat. No. 6,898,367, whichclaims priority to German patent applications 100 30 013.8 and 101 15487.9 and 101 15 589.1 and 101 15 486.0 and 101 15 488.7 and 101 15509.3 and 101 15 577.8 and 101 15 590.5, all of which are herebyincorporated by reference herein.

This application refers to U.S. patent application Ser. No. 09/881,048,now abandoned, entitled “ARRANGEMENT FOR STUDYING MICROSCOPICPREPARATIONS WITH A SCANNING MICROSCOPE”, which is incorporatedherewith.

This application refers to U.S. patent application Ser. No. 09/881,046,now U.S. Pat. No. 6,611,643, entitled “ILLUMINATING DEVICE ANDMICROSCOPE”, which is hereby incorporated by reference herein.

This application refers to U.S. patent application Ser. No. 09/880,825,now U.S. Pat. No. 6,567,164, entitled “ENTANGLED-PHOTON MICROSCOPE ANDCONFOCAL MICROSCOPE”, which is hereby incorporated by reference herein.

This application refers to U.S. patent application Ser. No. 09/881,062,now U.S. Pat. No. 6,888,674, entitled “ARRANGEMENT FOR EXAMININGMICROSCOPIC PREPARATIONS WITH A SCANNING MICROSCOPE, AND ILLUMINATIONDEVICE FOR A SCANNING MICROSCOPE”, which is hereby incorporated byreference herein.

This application refers to U.S. patent application Ser. No. 09/881,212,now U.S. Pat. No. 6,888,674, entitled “METHOD AND INSTRUMENT FORILLUMINATING AN OBJECT”, which is hereby incorporated by referenceherein.

This application refers to U.S. patent application Ser. No. 09/881,047,now U.S. Pat. No. 6,654,166, entitled “SCANNING MICROSCOPE WITHMULTIBAND ILLUMINATION AND OPTICAL COMPONENT FOR A SCANNING MICROSCOPEWITH MULTIBAND ILLUMINATION”, which is hereby incorporated by referenceherein.

This application refers to U.S. patent application Ser. No. 09/882,355,now U.S. Pat. No. 6,710,918, entitled “SCANNING MICROSCOPE”, which ishereby incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to a method for illuminating an object. Theinvention also relates to an instrument for illuminating an object.

BACKGROUND OF THE INVENTION

Laid-open patent specification DE 198 53 669 A1 discloses anultrashort-pulse source with controllable multiple-wavelength output,which is used especially in a multiphoton microscope. The system has anultrashort-pulse laser for producing ultrashort optical pulses of afixed wavelength and at least one wavelength conversion channel.

U.S. Pat. No. 6,097,870 discloses an arrangement for generating abroadband spectrum in the visible and infrared spectral range. Thearrangement is based on a microstructured fibre, into which the lightfrom a pump laser is injected. The pump light is broadened in themicrostructured fibre by non-linear effects. So-called photonic band gapmaterial or “photonic crystal fibres”, “holey fibres” or“microstructured fibres” are also employed as microstructured fibres.Configurations as a so-called “hollow fibre” are also known.

Another arrangement for generating a broadband spectrum is disclosed inthe publication by Birks et al.: “Supercontinuum generation in taperedfibres”, Opt. Lett. Vol. 25, p. 1415 (2000). A conventional opticalfibre having a fibre core, which has a taper at least along asubsection, is used in the arrangement. Optical fibres of this type areknown as so-called “tapered fibres”.

An optical amplifier, whose gain can be adjusted as a function of thewavelength, is known from the PCT application with the publicationnumber WO 00/04613. The said publication also discloses a fibre lightsource based on this principle.

Arc lamps are known as broadband light sources, and are employed in manyareas. One example is the U.S. Pat. No. 3,720,822 “XENON PHOTOGRAPHYLIGHT”, which discloses a xenon arc lamp for illumination inphotography.

Especially in microscopy, endoscopy, flow cytometry, chromatography andlithography, universal illuminating devices with high luminance areimportant for the illumination of objects. In scanning microscopy, asample is scanned with a light beam. To that end, lasers are often usedas the light source. For example, an arrangement having a single laserwhich emits several laser lines is known from EP 0 495 930: “KonfokalesMikroskopsystem für Mehrfarbenfluoreszenz” [confocal microscope systemfor multicolour fluorescence]. Mixed gas lasers, especially ArKr lasers,are mainly used for this at present. Examples of samples which arestudied include biological tissue or sections prepared with fluorescentdyes. In the field of material study, illumination light reflected fromthe sample is often detected. Solid-state lasers and dye lasers, as wellas fibre lasers and optical parametric oscillators (OPOs), upstream ofwhich a pump laser is arranged, are also frequently used.

Microspot arrays or so-called microplates are used in genetic, medicaland biodiagnosis for studying large numbers of specifically labelledspots, which are preferably applied in a grid. A microplate reader whichcan be adjusted both in excitation wavelength and in detectionwavelength is disclosed in the European Patient Application EP 0 841 557A2.

The illumination methods and illuminating instruments known from theprior art have several disadvantages. The known broadband illuminatinginstruments mostly have a low luminance compared with laser-basedilluminating devices, whereas the latter provide the user only withdiscrete wavelength lines whose spectral position and width can beadjusted only to a small extent, if at all. Owing to this limitation ofthe working spectrum, the known illuminating devices are not flexiblyusable. Laser-based illuminating devices and illuminating methods alsohave the disadvantage that, owing to the high coherence of the laserlight, disruptive interference phenomena, such as e.g. diffraction ringsand Newton's rings, occur. To reduce these interference effects,additional optical elements are often used, which reduce the light powerby intrinsic absorption and by scattering.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for illuminating anobject which is universally usable and flexible, furthermore provides abroad wavelength spectrum together with a high luminance, and alsominimizes interference phenomena.

The present invention provides a method for illuminating an objectcomprising the following steps:

-   -   generating a light beam with a laser,    -   injecting a light beam into a microstructured optical element        which spectrally broadens the light of the light beam,    -   shaping the spectrally broadened light beam to form an        illumination light beam, and    -   directing the illumination light beam onto the object.

It is another object of the present invention to provide an instrumentfor illuminating an object, which is universally usable and flexible,furthermore provides a broad wavelength spectrum together with a highluminance, and also minimizes interference phenomena.

The present invention provides an Illuminating instrument comprising: alaser that emits a light beam, a microstructured optical element thatspectrally broadens the light from the laser and an optical means forshaping the spectrally broadened light into an illumination light beam.

It is another object of the present invention to provide a device for amicroscopic inspection of an object, which is universally usable andflexible, furthermore provides a broad wavelength spectrum together witha high luminance, and also minimizes interference phenomena.

The present invention provides a device comprising: a laser that emits alight beam, a microstructured optical element that spectrally broadensthe light from the laser and an optical means for shaping the spectrallybroadened light into an illumination light beam.

The invention has the advantage that it is universally usable, easy tohandle and flexible, and furthermore provides illumination with lightfrom a wide wavelength range. The light also has very low coherence, sothat disruptive interference phenomena are avoided.

By using microstructured fibres, as described in the previouslymentioned U.S. Pat. No. 6,097,870 or in the publication by Birks et al.,a broad continuous wavelength spectrum is accessible. Arrangements ofthe disclosed type, however, are difficult to handle, inflexible andsusceptible to interference, especially because of the complexity of theindividual optical components and their relative adjustment.

A configuration variant in which a lens, which shapes the spectrallybroadened light into a beam, is arranged downstream of themicrostructured optical element, is especially advantageous. This lensis preferably located inside a casing which houses the entireinstrument, immediately in front of or in a light exit opening. The lensis preferably a variable lens with which various divergent, collimatedor convergent beam shapes can be produced.

All common laser types may be used as the laser. In a configuration, thelaser is a short-pulse laser, for example a mode-locked solid-statelaser, which emits light pulses with a pulse width of from 100 fs to 10ps. The wavelength of the laser is preferably matched to the “zerodispersion wavelength” of the fibre, or vice versa. Apparently, the zerodispersion wavelength can be “shifted” over a certain wavelength range,and this needs to be taken into account when pulling the fibre.

An embodiment of the illuminating device contains an instrument forvarying the power of the spectrally broadened light. In this case, it isadvantageous to configure the illuminating device in such a way that thepower of the spectrally broadened light can be varied or can be fullystopped-out with respect to at least one selectable wavelength or atleast one selectable wavelength range.

An instrument for varying the power of the spectrally broadened light ispreferably provided. Examples are acousto-optical or electro-opticalelements, such as acousto-optical tunable filters (AOTFs). It islikewise possible to use dielectric filters or colour filters, which arepreferably arranged in cascade. Particular flexibility is achieved ifthe filters are fitted in revolvers or in slide mounts, which allow easyinsertion into the beam path of the spectrally broadened light.

A configuration which makes it possible to select at least onewavelength range from the spectrally broadened light, the light of theselected wavelength range being directed onto the object, isadvantageous. This can be done, for example, using an instrument whichspectrally resolves the spectrally broadened light in a spatial fashion,in order to make it possible to suppress or fully stop-out spectralcomponents with a suitable variable aperture arrangement or filterarrangement, and subsequently recombine the remaining spectralcomponents to form a beam. A prism or a grating, for example, may beused for the spatial spectral resolution.

In a special configuration, the method according to the inventioncomprises the further step of adjusting the power of the spectrallybroadened light. To vary the power of the spectrally broadened light, inanother alternative embodiment, a Fabry-Perot filter is provided. LCDfilters can also be used.

In a configuration variant, the illuminating method comprises theadditional step of adjusting the spectral composition of the spectrallybroadened light.

An embodiment which directly has an operating element for adjusting thelight power and the spectral composition of the spectrally broadenedlight, is especially advantageous. This may be a control panel or a PC.The adjustment data is preferably transmitted in the form of electricalsignals to the illuminating instrument, or to the instrument for varyingthe power of the spectrally broadened light. Adjustment using sliders,which are displayed on a PC monitor and, for example, can be operatedusing a computer mouse, is particularly clear.

According to the invention, it has been discovered that the divergenceof the light injected into the microstructured optical element has aconsiderable influence on the spectral distribution of the spectrallybroadened light. In a flexible configuration, the illuminatinginstrument contains a focusing lens which focuses the light beam fromthe laser onto the microstructured optical element. Embodiment of thefocusing lens as a variable lens, for example as a zoom lens, isadvantageous.

Since the spectral distribution of the spectrally broadened lightdepends on the polarization and the wavelength of the light injectedinto the microstructured optical element, in a particular configuration,instruments are provided for adjusting and influencing these parameters.In the case of lasers, which emit linearly polarized light, a rotatablymounted λ/2 plate is used to rotate the polarization plane. Somewhatmore elaborate, but also more flexible, is the use of a Pockels cell,which also makes it possible to set any desired elliptical polarization,or of a Faraday rotator. To adjust the wavelength, a birefringent plateor a tiltable etalon is preferably provided in the laser.

In a particular configuration, an instrument is provided which permitsanalysis of the broadened-wavelength light, in particular with regard tothe spectral composition and the luminance. The analysis instrument isarranged in such a way that part of the spectrally broadened light issplit off, for example with the aid of a beam splitter, and fed to theanalysis instrument. The analysis instrument is preferably aspectrometer. It contains, for example, a prism or a grating for thespatial spectral resolution, and a CCD element or a multichannelphotomultiplier as the detector. In another variant, the analysisinstrument contains a multiband detector. Semiconductor spectrometerscan also be employed.

To establish the power of the spectrally broadened light, the detectorsare configured in such a way that an electrical signal, which isproportional to the light power and can be evaluated by electronics or acomputer, is generated.

The embodiment which contains a display for the power of the spectrallybroadened light and/or for the spectral composition of the spectrallybroadened light is advantageous. The display is preferably fitteddirectly on the casing or to the control panel. In another embodiment,the monitor of a PC is used for displaying the power and/or the spectralcomposition.

In another configuration, the method according to the inventioncomprises the step of adjusting the polarization of the spectrallybroadened light. To that end, a rotatably arranged polarization filter,a λ/2 plate, a Pockels cell or a Faraday rotator is provided.

In an embodiment, the laser is a pulse laser which preferably emitslight pulses with a pulse energy in excess of 1 nJ. In relation to thisconfiguration, the method according to the invention comprises theadditional step of adjusting the pulse width of the spectrally broadenedlight. It is furthermore advantageous that the method allows the furtherstep of adjusting the chirp of the spectrally broadened light. Usingthese additional steps, the pulse properties of the light directed ontothe object can be matched individually to the object in question.“Chirp” means the time sequence of the light are different wavelengthswithin a pulse. To that end, the instrument according to the inventionpreferably comprises a prism or a grating arrangement which, in aconfiguration, is combined with an LCD strip grating. Arrangements forvarying the pulse width and the chirp are adequately known to a personskilled in the art.

The illuminating method and instrument can be used, particularly toilluminate a microscopic object, in particular in a microscope, a videomicroscope, a scanning microscope or confocal scanning microscope. It isadvantageous if the wavelength of the light directed onto the object, inthe case of fluorescence applications or applications which are based onForster transfer, is matched accurately to the excitation wavelength ofthe fluorochromes present in the object.

The illuminating method and instrument can also be used advantageouslyin endoscopy, flow cytometry and lithography.

In a configuration of the scanning microscope, the microstructuredoptical element is constructed from a plurality of micro-opticalstructure elements, which have at least two different optical densities.A configuration in which the optical element contains a first region anda second region, the first region having a homogeneous structure and amicrostructure comprising micro-optical structure elements being formedin the second region. It is furthermore advantageous if the first regionencloses the second region. The micro-optical structure elements arepreferably cannulas, webs, honeycombs, tubes or cavities.

In another configuration, the microstructured optical element consistsof adjacent glass or plastic material and cavities. In an alternativeembodiment, the microstructured optical element consists of photonicband gap material and is configured as an optical fibre. An opticaldiode, which suppresses back-reflections of the light beam due to theends of the optical fibre, is preferably arranged between the laser andthe optical fibre.

An alternative embodiment, which is simple to implement, contains aconventional optical fibre having a fibre core diameter of approximately9 μm, which has a taper at least along a subsection, as themicrostructured optical element. Optical fibres of this type are knownas so-called “tapered fibres”. The optical fibre preferably has anoverall length of 1 m and a taper over a length of from 30 mm to 90 mm.The diameter of the optical fibre, in a configuration, is approximately2 μm in the region of the taper. The fibre core diameter iscorrespondingly in the nanometer range.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject-matter of the invention is diagrammatically represented inthe drawing and will be described below with the aid of the figures, inwhich:

FIG. 1 shows a flow chart of the method according to the invention,

FIG. 2 shows an illuminating device according to the invention with apower meter and a display,

FIG. 3 shows, as an example, the use of an instrument according to theinvention in a confocal scanning microscope,

FIG. 4 shows an embodiment of the microstructured optical element, and

FIG. 5 shows another embodiment of the microstructured optical element.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a flow chart of the method according to the invention. In afirst step, the light from a laser is injected 1 into a microstructuredoptical element that spectrally broadens the light. In this case, thelight is guided to the microstructured optical element, for example withthe aid of mirrors, and is preferably focused onto the microstructuredoptical element using a variable lens. In a second step, the lightemerging from the microstructured optical element is shaped 3 to form anillumination light beam, preferably with the aid of optical means whichare configured as lens systems. In a further step, the illuminationlight beam is directed 5 onto the object.

FIG. 2 shows an illuminating instrument 7 which contains a laser 9 thatis embodied as a mode-locked Ti:sapphire laser 11 and emits a light beam13, which is shown in dashes, with the property of an optical pulsetrain. The width of the light pulses is approximately 100 fs with arepetition rate of approximately 80 MHz. The light beam 13 is focused bythe optical means 15, which is configured as a zoom lens 17 and isarranged displaceably along the propagation direction of the light beam,onto a microstructured optical element 19. The microstructured opticalelement 19 consists of an optical fibre 23 having a taper 21. In themicrostructured optical element, the light from the laser is spectrallybroadened. All the components are located in a casing 25 having a lightexit opening 27, through which the illumination light beam 29 leaves thecasing 25 as a divergent beam. The spectrum of the spectrally broadenedlight 31 extends from approximately 300 nm to 1600 nm, the light powerbeing substantially constant over the entire spectrum. The spectrallybroadened light 31 emerging from the optical fibre 23 is shaped with theaid of the lens 33 to form the collimated illumination light beam 29.Using the beam splitter 35, a subsidiary light beam 37 of theillumination light beam 29 is split off and diverted onto an analysisinstrument 39. The latter contains a prism 41 which spectrally spreadsthe subsidiary light beam 37 in a spatial fashion to form a light cone43 that diverges in the spreading plane, and a photodiode linear array45 for detecting the light. The photodiode linear array 45 generateselectrical signals, which are proportional to the power of the light ofthe spectral range in question and are fed to a processing unit 47. Thelatter is connected to a PC 49, on whose monitor 51 the spectralcomposition is displayed in the form of a graph 53 within a coordinatesystem having two axes 55, 57. The wavelength is plotted against theaxis 55 and the power of the light is plotted against the axis 57.Clicking the graph 53 using a computer mouse 59 and moving the computermouse 59 at the same time generates a dotted graph 61, which can bedeformed in accordance with the movement of the computer mouse 59. Assoon as the computer mouse 59 is clicked again, the computer 49 drivesan instrument for varying the power 63 in such a way as to produce thespectral composition preselected by the dotted graph 61. The instrumentfor varying the power 63 of the spectrally broadened light 31 isdesigned as an AOTF 65 (acousto-optical tunable filter), and isconfigured in such a way that the wavelengths are influencedindependently of one another, so that the spectral composition of thespectrally broadened light 31 can be adjusted. A system for controllingthe output power of the laser 9 by means of the computer is furthermoreprovided. The user makes adjustments with the aid of the computer mouse59. A slider 67, which is used for adjusting the overall power of thespectrally modified light 31, is represented on the monitor 51.

FIG. 3 represents, as an example, the use of an instrument according tothe invention in a confocal scanning microscope 69. The illuminationlight beam 29 coming from the illuminating instrument 7 is reflected bya beam splitter 71 to the scanning module 73, which contains acardan-suspended scanning mirror 75 that guides the light beam 29through the microscope lens 77 and over or through the object 79. In thecase of non-transparent objects 79, the illumination light beam 29 isguided over the object surface. In the case of biological objects 79 ortransparent objects 79, the illumination light beam 29 can also beguided through the object 79. This means that various focal planes ofthe object 79 are illuminated successively by the illumination lightbeam 29, and are hence scanned. Subsequent combination then gives athree-dimensional image of the object 79. The light beam 29 coming fromthe illuminating instrument 7 is represented in the figure as a solidline. The light 81 leaving the object 79 travels through the microscopelens 77 and, via the scanning module 73, to the beam splitter 71, thenit passes through the latter and strikes the detector 83, which isembodied as a photomultiplier. The light 81 leaving the object 79 isrepresented as a dashed line. In the detector 83, electrical detectionsignals proportional to the power of the light 81 leaving the object 79are generated and processed. The illumination pinhole 85 and thedetection pinhole 87, which are normally provided in a confocal scanningmicroscope, are indicated schematically for the sake of completeness.For better clarity, however, a few optical elements for guiding andshaping the light beams are omitted. These are adequately known to aperson skilled in this field.

FIG. 4 shows an embodiment of the microstructured optical element 19. Itconsists of photonic band gap material, which has a special honeycombedmicrostructure 89. The honeycombed structure that is shown isparticularly suitable for generating broadband light. The diameter ofthe glass inner cannula 91 is approximately 1.9 μm. The inner cannula 91is surrounded by glass webs 93. The glass webs 93 form honeycombedcavities 95. These micro-optical structure elements together form asecond region 97, which is enclosed by a first region 99 that isdesigned as a glass cladding.

FIG. 5 schematically shows an embodiment of the microstructured opticalelement 19. In this embodiment, the microstructured optical element 19consists of conventional optical fibre 101 having an external diameterof 125 μm and a fibre core 103, which has a diameter of 6 μm. In theregion of a 300 mm long taper 105, the external diameter of the opticalfibre 101 is reduced to 1.8 μm. In this region, the diameter of thefibre core 103 is then only fractions of a micrometer.

The invention has been described with reference to a particularembodiment. It is, however, obvious that modifications and amendmentsmay be made without thereby departing from the scope of protection ofthe following claims.

1. A microscope comprising: a light source configured to emit anilluminating light beam for illumination of a specimen; a beam splitterconfigured to separate measuring light out of the illuminating lightbeam; and a power detection apparatus configured to determine a lightpower level of the illuminating light beam, the power detectionapparatus being configured to receive the measuring light and includinga light detection apparatus configured for simultaneous color-selectivedetection of the measuring light.
 2. The microscope as defined in claim1 wherein the apparatus for simultaneous color-selective detectionincludes a spatial spectral spreading element.
 3. The microscope asdefined in claim 2 wherein the spatial spectral spreading element is aprism.
 4. The microscope as defined in claim 1 wherein the apparatus forsimultaneous color-selective detection comprises at least one detectorconfigured to receive the measuring light.
 5. The microscope as definedin claim 4 wherein the at least one detector includes a plurality ofindividual detectors configured to each receive spectrally differentcomponents of the measuring light.
 6. The microscope as defined in claim4 wherein the at least one detector includes at least one of aphotodiode, a photomultiplier, a photodiode row, a photodiode array, aCCD element, a photomultiplier array and a photomultiplier row.
 7. Anapparatus for determining a light power level of an illuminating lightbeam, the apparatus comprising: a beam splitter configured to separatemeasuring light out of the illuminating light beam; and a detectiondevice configured for simultaneous color-selective detection of themeasuring light.
 8. The apparatus as defined in claim 7 wherein thedevice for simultaneous color-selective detection includes a spatialspectral spreading element.
 9. The apparatus as defined in claim 8wherein the spatial spectral spreading element is a prism.
 10. Theapparatus as defined in claim 7 wherein the device for simultaneouscolor-selective detection includes at least one detector configured toreceive the measuring light.
 11. The apparatus as defined in claim 10wherein the at least one detector includes a plurality of individualdetectors that each receive spectrally different components of themeasuring light.
 12. The apparatus as defined in claim 10 wherein the atleast one detector includes at least one of a photodiode, aphotomultiplier, a photodiode row, a photodiode array, a CCD element, aphotomultiplier array and a photomultiplier row.
 13. A method fordetermining a light power level of an illuminating light beam, themethod comprising: emitting, using a light source, an illuminating lightbeam for illumination of a specimen; separating measuring light out ofthe illuminating light beam using a beam splitter; and simultaneouslyand color-selectively detecting the measuring light using a lightdetection apparatus.
 14. The method as defined in claim 13 furthercomprising generating electrical signals each proportional to arespective power level of respective spectral components of the measuredlight.
 15. The method as defined in claim 13 wherein the light detectionapparatus includes a spatial spectral spreading element.
 16. The methodas defined in claim 15 wherein the spatial spectral spreading element isa prism.
 17. The method as defined in claim 13 wherein the lightdetection apparatus includes a at least one detector configured toreceive the measuring light.
 18. The method as defined in claim 17wherein the at least one detector includes a plurality of individualdetectors configured to each receive spectrally different components ofthe measuring light.
 19. The method as defined in claim 17 wherein theat least one detector includes at least one of a photodiode, aphotomultiplier, a photodiode row, a photodiode array, a CCD element, aphotomultiplier array and a photomultiplier row.